Advancement of gene therapy to the clinic is hampered by the lack of efficient and reliable means of gene delivery. Efficiency of gene transfer mediated by non-viral gene delivery diseases such as hemophilia, it is highly important to maintain prolonged steady release of therapeutic genes. It is hypothesized that controlled and efficient long-term delivery of genes could be achieved by combining polymer-based controlled release devices with non-viral vectors. A very important issue to be addressed in developing clinically useful gene delivery systems is DNA stability. Inactivation of DNA due to physical and chemical degradation during preparation of controlled release systems, long-term storage, and following administration, severely compromises the efficacy of gene therapy. Understanding the degradation pathways of DNA should lead to rational design of effective strategies for DNA stabilization. The specific aims of this proposal are (1) to prepare and characterize biodegradable poly(lactic) acid co-glycolic acid) (PLGA) microsphere encapsulating plasmid DNA complexed with selected non-viral vectors including cationic polymers and peptides, (2) to evaluate the release of complexed plasmid DNA from PLGA microspheres and transfection efficiency in vitro, (3) to identify the non-enzymatic pathways involved in the degradation of plasmid DNA complexed with selected cationic vectors in dry and hydrated PLGA PLGA microspheres using robust analytical techniques, and (4) to design and test methods of plasmid DNA stabilization based on the knowledge gained from polymer-based controlled delivery systems for gene therapy.